Project Summary: Our long-term objective is to commercialize a fast, simple, flexible, and economical system for affinity-tag protein purification and tag removal. This technology was developed as a result of NIH-funded studies on the folding, stability and enzymology of the Bacillus protease subtilisin. The transformative purification technology combines three components: 1) a regulated, highly-specific protease (Psub) that is constitutively inactive; 2) a small molecule (imidazole) that activates the Psub; 3) a high-affinity inhibitor (Pro). Immobilized Psub (in the off-state) can capture a Pro-fusion protein from a cellular extract, allowing contaminants to be washed away. Subsequent addition of imidazole to the immobilized complex releases pure, tag-free target protein and leaves the Pro-tag tightly bound to Psub. Recombinant proteins are frequently fused with other proteins or peptides to facilitate expression and purification. The tags enable target protein binding in affinity purification, but ultimately must be processed by a site-specific protease. Tag removal, however, is frequently expensive, inefficient, and sometimes problematic. The technical innovation of our system is the integration of seamless tag removal into the purification process. This provides simplicity, efficiency, and robustness that is not available in any other system. Improved understanding of protein engineering principles has resulted in highly-efficient Psubs and Pro affinity tags. We will develop and test purification materials and methods around imidazole-triggered chemistry and improved affinity tags. The Specific Aims are: 1) produce imidazole-triggered Psubs at low cost; 2) clone a range of test proteins into expression vectors with the new tags; 3) Measure soluble expression for the Pro-tagged test proteins; 4) Develop and test purification methods using Psubs and test proteins chosen to challenge purification methods; 5) Refine Psubs if needed by screening phage libraries. The system eventually should be suitable for both high- throughput, purification of proteins on the laboratory scale, as well as process-scale purification of pharmaceutical proteins. Better purification technology should advance in all areas of biology.